Enzyme-linked immunosorbent assay for immunoglobulin G and M antibodies to Chlamydia trachomatis in human sera.

Microimmunofluorescence methods used for detection of immunoglobulin G and M antibodies to Chlamydia trachomatis are not available to many clinical laboratories. We evaluated a simple enzyme-linked immunosorbent assay in which serotype L2 elementary bodies are used as antigen. The enzyme-linked immunosorbent assay proved satisfactory for the detection of serum IgG. A total of 160 human sera were tested, and the results correlated well with those obtained by microimmunofluorescence. Results for IgM antibody detection were not as successful, and correlation with current methods was poor.     

Enzyme-linked immunosorbent assay (ELISA) for mumps virus antibodies

An ELISA for the detection of mumps virus-specific IgG and IgM antibodies was developed. Three different antigen preparations were compared. Equally good results were obtained with virus concentrated by ultracentrifugation and with virus that was further purified by sucrose gradient centrifugation. Crude infected allantoic fluid was unsuitable for use as antigen. The variability and reproducibility of the tests were satisfactory. When the ELISA was compared to conventional serological methods, a good correlation of IgG absorbance values with complement fixation (CF) antibody titers was found (r = 0.574), the ELISA being more sensitive in detecting antibodies in acute-phase sera. For the determination of immunity, ELISA IgG values were compared with results obtained in hemagglutination-inhibition (HI) and hemolysis-in-gel (HIG) tests. Again there was a good correlation with both tests (r_HI = 0.528, r_HIG = 0.667). The ELISA was more sensitive than the HI and HIG test for the detection of low levels of antibodies.     

Enzyme-linked immunosorbent assay for immunoglobulin G and immunoglobulin A antibodies to Shigella flexneri antigens.

An enzyme-linked immunosorbent assay has been developed to detect class-specific antibodies to Shigella flexneri lipopolysaccharide antigens. This enzyme-linked immunosorbent assay system has been used to measure antibodies present in serum or intestinal secretions without further purification. It is considerably more sensitive than passive hemagglutination, allowing detection of as little as 1.3 ng of specific immunoglobulin G antibody per ml in immune sera. Optimal conditions for this assay are outlined in this report.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin A and G antibodies toCampylobacter pylori

An enzyme-linked immunosorbent assay (ELISA) employing an acid-glycine extract was used to detect IgG and IgA antibodies toCampylobacter pylori in sera from 179 patients with upper gastrointestinal disease, 174 blood donors and 65 children. The incidence of positive ELISA results clearly increased with the severity of histopathologic findings in the antrum mucosa and was also high in patients with peptic ulcers and gastric cancer. The incidence in blood donors and children was much lower and increased with age. The results achieved with the ELISA were similar to those observed previously using the immunoblot method. Differences between whole cell preparation and acid-glycine extract with respect to their protein profiles and immunoblot reactivities were minor. IgM titres were very low and could not be related to histopathological findings, peptic lesions or culture findings. The ELISA may be particularly useful for monitoring the outcome of therapy aimed at eradication ofCampylobacter pylori.     

Mumps-specific immunoglobulin M and G antibodies in natural mumps infection as measured by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay for the demonstration of mumps immunoglobulin G (IgG ELISA) and immunoglobulin M antibodies (IgM ELISA) in serum was compared with complement fixation (CF), hemagglutination inhibition (HI), and hemolysis-in-gel (HIG) tests. The antibody levels measured by IgG ELISA had a high positive correlation with the CF and HIG tests, whereas only a moderate correlation was found between IgG ELISA and HI. Similar patterns of antibody response were observed with IgG ELISA, CF, and HIG: the antibody titres increased rapidly after the onset of symptoms and reached the maximal values in about three weeks. The HI antibodies developed more slowly during the first week of disease, after which the titres increased rapidly up to the fourth week. IgM antibodies measured by ELISA developed soon after onset of symptoms; most patients had IgM antibodies from the second day, and the highest titres were reached within the first week. The antibody response in mumps parotitis did not differ from that in mumps meningitis/encephalitis, while relatively higher antibody titres were found in patients with orchitis/epididymitis. The diagnostic efficiencies of the methods were compared with serum specimens from 33 patients who had a serologically verified mumps infection by at least one of the five methods used (rising antibody titres in paired sera or detectable IgM): IgM ELISA detected all 33 cases, IgG ELISA 29, HIG 28, HI 23, and CF 13. In 27 cases, IgM antibodies were already present in the acute phase serum specimens. It was concluded that mumps IgM ELISA is a more rapid and sensitive means for the serological diagnosis of mumps infection than the conventional tests.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin G and M antibodies to teichoic acid in intravascular staphylococcal disease

An enzyme-linked immunosorbent assay (ELISA) for detection of IgG and IgM antibodies to cell-wall teichoic acids ofStaphylococcus aureus and three defined coagulase-negative staphylococci was tested using serum samples from 11 cases of intravascular coagulasenegative staphylococcal infections, 13 cases ofStaphylococcus aureus endocarditis, and 24 patients with no evidence of infection. IgG antibody titers to all four teichoic acids in the 13 patients withStaphylococcus aureus endocarditis were significantly different from those in noninfected control patients (p<0.0001). In contrast, IgG antibody titers in serum from 11 cases of intravascular coagulase-negative Staphylococcal infection were not significantly different from those in control sera. There were no differences in IgM antibody titers of the three groups. Although the ELISA was sensitive in detectingStaphylococcus aureus endocarditis, it was not reliable in the detection of intravascular coagulasenegative Staphylococcal infections, even when tested with specific teichoic acid.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Escherichia coli Vero cytotoxin 1.

The frequency of Vero cytotoxin 1 (VT1)-neutralizing antibody (NAb) in serum specimens from 790 age-stratified (0 to 70 years) control individuals from Toronto was 61 of 790 (7.7%), with a peak of 19% in the 20- to 30-year-old age group and a second peak of 16.7% in the 60- to 70-year-old age group. A total of 568 serum specimens, including 538 from the 790 Toronto control subjects, 21 from patients from three outbreaks of VT-producing Escherichia coli (VTEC) infection, and 9 known VT1-NAb-positive serum specimens from patients with hemolytic-uremic syndrome (HUS), were then tested for the presence of anti-VT1 immunoglobulin G (IgG) by an enzyme-linked immunosorbent assay (ELISA). The mean ELISA values of 522 VT1-NAb-negative serum specimens and 46 VT1-NAb-positive serum specimens were 0.09 +/- 0.06 (range, 0 to 0.56) and 0.78 +/- 0.66 (range, 0.16 to 2.91), respectively (P < 0.001; Student's t test). With a breakpoint of 0.21 (mean ELISA value of the VT1-NAb-negative sera + 2 standard deviations), the sensitivity, specificity, positive predictive value, and negative predictive value of the VT1 IgG ELISA compared with those of the VT1-NAb assay were, respectively, 95.7, 98.7, 86.3, and 99.6%. There were nine discrepant serum specimens, of which seven were anti-VT1 IgG positive and VT1-NAb negative and two were anti-VT1 IgG negative and VT1-NAb positive. The ELISA was also used for testing 238 control serum specimens from The Netherlands, Japan, and India and acute- and convalescent-phase serum specimens from 42 Toronto patients with HUS. The frequencies of anti-VT1 IgG (with VT1-NAb frequencies in parantheses) in control sera from the Netherlands, Japan, and India were 6% (3%), 1.1% (0%), and 12% (10%), respectively, with no age clustering. The frequencies of anti-VT1 IgG seropositivity in HUS patients were 5 of 14 (35.7%) in patients with unknown toxin exposure, 2 of 22 (9.1%) in individuals with known exposure to VT1 plus VT2 or VT1 alone, and 0 of 6 (0%) in patients exposed to only VT2. Development of serum anti-VT1 IgG response appears to be the exception rather than the rule in sporadic HUS patients infected with VTEC expressing VT1. However, in two family outbreaks associated with VTEC strains expressing VT1 alone and VT1 plus VT2, respectively, the presence of anti-VT1 IgG in virtually all exposed individuals who remained symptom free suggests that the presence of antibody was associated with protection.     

Enzyme-linked immunosorbent assay for streptococcal M protein antibodies.

The enzyme-linked immunosorbent assay (ELISA) described by Engvall and Perlmann, which uses antigen-coated tubes and enzyme-labeled anti-immunoglobulin, has been used for the detection of antibodies against streptococcal M protein. The antigen used in the assay was obtained by guanidine extraction of type M-12 streptococcal cell walls followed by hydroxyapatite chromatography. This antigen has the capacity to elicit bactericidal antibodies in rabbits. The results show that the ELISA is specific and highly sensitive for the detection of antibodies in rabbit and human antisera. Preliminary results suggest that, when M-12 antigen is used, the antibodies detected by ELISA are the same antibodies detected in the bactericidal test. The assay has been performed with human and rabbit sera. There was a 96% agreement between bactericidal and ELISA results with rabbit sera and 97.5% agreement with human sera. All bactericidal antibody-positive sera tested thus far yielded positive ELISA results.     

Enzyme-linked immunosorbent assay for immunoglobulin G antibody to encephalomyocarditis virus

An enzyme-linked immunosorbent assay for immunoglobulin G antibody to encephalomyocarditis virus was developed. This assay was comparable to antibody assay by neutralization. Its adaptability should be useful for laboratory and epidemiological studies of infections due to encephalomyocarditis virus.     

Enzyme-linked immunosorbent assay for detection ofMycoplasma pneumoniae specific immunoglobulin M

An ELISA was developed for the detection of IgM antibodies to Mycoplasma pneumoniae in human sera, using microtiter plates coated with rabbit antiserum to human IgM selecting for IgM antibodies in the first reaction step. The specific antibodies were detected using enzyme-labelled, detergent-solubilized antigen. The complement fixation test was used as reference method. In a prospective study of 59 patients with community-acquired pneumonia, 13 of whom had evidence of mycoplasmal etiology, the ELISA was shown to have high specificity (97%). In samples taken seven days after onset of the disease all complement-fixation positive samples (n = 20) but one were positive, demonstrating the diagnostic value of a positive test in samples taken after that period.     

Microenzyme-linked immunosorbent assay for detection of immunoglobulin G and immunoglobulin M antibodies to Legionella pneumophila

The microenzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin M and G (IgM, IgG) antibodies to Legionella pneumophila serogroup 1 antigens was evaluated. IgM antibodies were measured by both double-sandwich and single-sandwich techniques. These assays were compared with the previously standardized indirect immunofluorescence test in four groups of subjects: (i) pneumonia patients with culture-proven Legionnaires disease with serogroup 1 isolates, (ii) pneumonia patients with serogroup 1 organisms detected by direct immunofluorescence testing of respiratory secretions but without culture confirmation, (iii) pneumonia patients with negative culture and direct immunofluorescence tests, and (iv) healthy hospital employees. In addition, the sensitivity and specificity of the IgG ELISA were evaluated with larger groups of controls and Legionnaires disease patients. The ELISA was more sensitive than the indirect immunofluorescence test. However, it detected antibody rises in pneumonia patients without culture or direct immunofluorescence evidence of L. pneumophila serogroup 1 infection, thereby suggesting that the specificity of the ELISA was slightly lower than that of the indirect immunofluorescence test. The double-sandwich ELISA was a sensitive method for detecting IgM antibodies and, as previously reported, appeared to be free from interference by rheumatoid factor. IgM anti-Legionella antibodies detected by the ELISA appeared earlier and were less persistent than IgG antibodies. In addition, the IgM ELISA was useful in detecting antibodies in necropsy serum samples obtained from patients dying acutely of Legionnaires disease. The data presented show that the ELISA is a reliable method for the detection of specific anti-Legionella antibodies.     

Detection of antichlamydial immunoglobulin G and M antibodies by enzyme-linked immunosorbent assay

Chlamydia trachomatis causes a wide range of infections in adults and conjunctivitis and pneumonia in neonates. The complement fixation test for chlamydial antibody is broadly reactive, but possesses low sensitivity, whereas the microimmunofluorescence test is highly sensitive, but technically difficult to perform. A simple, rapid enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of immunoglobulin G (IgG) and IgM antibodies to C. trachomatis. Wells of microtiter plates were coated with Renografin-purified elementary bodies (serotype L2) grown in cycloheximide-treated McCoy cells, and serum antibody was detected with peroxidase-labeled goat antihuman IgG and IgM antibody. Of 41 sera tested from patients with lymphogranuloma venereum, pelvic inflammatory disease, cervicitis, or urethritis there was a 90 and 63% correlation of positive results for IgG and IgM, respectively, by microimmunofluorescence and ELISA. Of the positive correlates, ELISA titers were up to 128 times higher than microimmunofluorescence titers for IgG and IgM. The ELISA detected no false-positive results, but missed two positive results for IgG. Both of these sera were reactive against serotypes C and J, suggesting that the ELISA with LGV L2 antigen may not measure antibodies to serotypes within the C serogroup. The IgM ELISA detected 7 negative and 4 positive results not detected by the microimmunofluorescence test. Of four paired sera examined by ELISA, three showed a fourfold rise in IgG antibody titer, and one showed a twofold rise. Further evaluation of this ELISA will be required to determine how useful it will be in seroepidemiological studies and as a diagnostic tool.     

Enzyme-linked immunosorbent assay for detection of antibodies to influenza A and B and parainfluenza type 1 in sera of patients.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to influenza A/Hong Kong/1/68, influenza A/Victoria/3/75, influenza B/Hong Kong/5/72, and parainfluenza type 1 viruses. Development and standardization of the method indicated that an acetone-ethyl alcohol mixture was a suitable fixative for the preparation of the solid-phase coupled antigen. The addition of sodium azide to the enzyme-conjugated solution and the concentrations of the enzyme-conjugate antiglobulin and test sera employed were all critical factors in the success of the ELISA procedure. The ELISA test was specific; there was no cross-reaction between influenza A and B or parainfluenza type 1 viruses. The concordance between ELISA and hemagglutination inhibition results suggested that both tests probably detected the same type of antibodies. The ELISA procedure was 8 to 64 times more sensitive than complement fixation and/or hemagglutination inhibition tests. Low levels of antibody in patients' sera were detected only by the ELISA test. During the course of the testing period false positive reactions were not encountered. The results of ELISA could be obtained within 3 h. The ELISA test required a very small amount of serum and, therefore, offered an opportunity to detect the presence of maternal antibodies to influenza viruses in blood collected from infants by heel prick.     

Enzyme-linked immunosorbent assay for chlamydial antibodies.

An enzyme-linked immunosorbent assay (ELISA) detected chlamydial antibodies in human sera. The assay antigen produced in cell cultures infected with Chlamydia psittaci was Formalin-fixed to microplates. Single convalescent-phase sera positive for chlamydial antibodies by a complement-fixation test were positive at even higher dilutions by ELISA. Paired sera with diagnostic rises in complement-fixing antibody showed seroconversion by ELISA also. Control sera from persons with no history of chlamydial infection were negative by both tests. Sera from patients with psittacosis or lymphogranuloma venereum were ELISA positive, indicating that the assay with the antigen used in this study is genus specific rather than species specific.     

Enzyme-linked immunosorbent assay for immunoglobulin G antibody to Pasteurella multocida in rabbits.

Three antigen preparations of Pasteurella multocida, lipopolysaccharide antigen, boiled-cell extract antigen, and boiled whole-bacterium antigen, were used in an enzyme-linked immunosorbent assay (ELISA) to detect rabbit immunoglobulin G antibody to P. multocida. The sensitivity of each antigen preparation was compared by using sera from P. multocida-infected and uninfected rabbits and sera from two rabbits immunized with different serotypes of P. multocida. In the ELISA, all three antigen preparations detected high titers of antibodies in infected rabbits and markedly lower levels in uninfected rabbits. When whole-bacterium or boiled-cell extract antigens were used, the ELISA detected antibodies in sera from both immunized rabbits, but with lipopolysaccharide antigen, only antibody to the homologous serotype was detected. Sera absorbed with P. multocida and Bordetella bronchiseptica, another respiratory pathogen of rabbits, revealed that antibodies detected in the ELISA did not cross-react. Since the lipopolysaccharide antigen was more difficult to prepare and may be type specific, and since the whole-bacterium antigen was the least sensitive, the boiled-cell extract was chosen as the best antigen preparation to use in the ELISA.     

Enzyme-linked immunosorbent assay for immunoglobulin M specific antibody for the diagnosis of melioidosis.

Indirect hemagglutination (IHA) is commonly used for serodiagnosis of melioidosis. However, in endemic areas, high background titers in normal populations and occasional low titers in patients with septicemic melioidosis prompted a search for a more sensitive and more specific method of serodiagnosis. An indirect fluorescent-antibody test for immunoglobulin M (IgM) specific antibody to Pseudomonas pseudomallei was more sensitive and more specific, but fluorescence microscopes are rarely available in the endemic areas. An enzyme-linked immunosorbent assay (ELISA) for IgM antibody is an attractive alternative. An indirect ELISA for IgM antibody (IgM ELISA) and an IgM antibody capture ELISA for melioidosis were developed. Both tests, together with IHA, were evaluated for 153 serum specimens from blood donors and 16 serum specimens from 16 melioidosis patients. It was found that IHA, the IgM ELISA, and the IgM antibody capture ELISA had sensitivities of 88, 88, and 75%, respectively, with specificities of 97.4, 92.2, and 91.5%, respectively. When IHA was combined with IgM ELISA, a sensitivity of 100% and a specificity of 95.4% were obtained. The IgM ELISA and IHA should be used in combination for serodiagnosis of melioidosis.     

Enzyme-linked immunosorbent assay (ELISA) for detection of specific IgA antibodies to mumps virus

A sensitive enzyme-linked immunosorbent assay (ELISA) is described for detection of IgA antibodies to mumps virus. Specific mumps IgA antibodies could be demonstrated in 10 patients with mumps virus infections. No specific mumps IgA antibodies (titres <1/40) were detected by ELISA in 46 control sera (healthy adults; hospitalised patients with various other diseases). The potential application of the ELISA mumps IgA technique in serodiagnosis of mumps infections is discussed.     

Measurement of immunoglobulin G and M antibodies to type 3 pneumococcal capsular polysaccharide by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed to measure immunoglobulin G (IgG) and IgM type 3 antipneumococcal capsular polysaccharide antibodies. The use of Fab2 fragments of rabbit antipneumococcal IgG antibody in the antibody-antigen sandwich increased the sensitivity for measuring IgM antibodies and decreased background activity in antigen-free cuvettes. This methodology detected type 3 IgM antibody responses in six of six subjects vaccinated with polyvalent pneumococcal vaccine and detected type 3 IgG antibody responses in three subjects. Results of the enzyme-linked immunosorbent assay and radioimmunoassay procedures were concordant, and postvaccination enzyme-linked immunosorbent assay IgM titers showed a stronger correlation with total radioimmunoassay antibody than did postvaccination ELISA IgG titers.